Construction and application of TAGLN2 gene defect type mouse model

Abstract

The invention discloses construction and application of a TAGLN2 gene defect type mouse model, which utilizes a CRISPR / Cas9 gene editing technology, uses gRNA to guide Cas9 protein to respectively shear at specific sites at the upstream of a second exon and the downstream of a fifth exon of a TAGLN2 gene, and realizes TAGLN2 gene knockout. The mouse model constructed by the invention provides a basis for researching the molecular mechanism of the TAGLN2 gene, and is beneficial to researching the pathogenesis of metabolic diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical animal models, and specifically relates to TAGLN2 the construction and application of a gene-deficient mouse model. Background Art

[0002] As an important tool in biomedical research, the mouse model plays a key role in exploring disease mechanisms, drug development, and disease treatment. The mouse model can simulate the occurrence and development process of human diseases, helping scientists deeply understand the etiology and pathophysiological changes of diseases. Conducting drug tests on the mouse model can effectively evaluate the efficacy and safety of drugs.

[0003] On the other hand, transgelin-2 ( TAGLN2 gene) is an important actin cytoskeleton-binding protein and belongs to the transgelin superfamily; it is mainly localized in the cytoplasm of cells and can affect the cell phenotype by directly regulating the expression of related genes. TAGLN2 The TAGLN2 gene is highly expressed in smooth muscle cells and fibroblasts and is involved in the regulation of multiple cell functions, including angiogenesis, cytoskeleton remodeling, cell migration, apoptosis, and proliferation. In the immune response, the TAGLN2 gene plays an important role; it can regulate the activation of T cells and participate in the stabilization of T cell-B cell conjugates in B cells. In addition, TAGLN2 the gene is significantly upregulated in lipopolysaccharide-stimulated macrophages, promoting the phagocytosis of macrophages. Recombinant TAGLN2 gene protein can also enhance the function of dendritic cells to inhibit tumor growth and metastasis. TAGLN2 The expression of the TAGLN2 gene is closely related to the prognosis in multiple cancers. Studies have shown that TAGLN2 the high expression of the TAGLN2 gene is associated with poor prognosis in multiple cancers and is closely related to processes such as tumor proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT); for example, in clear cell renal cell carcinoma,

[0004] In view of TAGLN2 the important role played by the TAGLN2 gene in multiple physiological and pathological processes, constructing a TAGLN2The mechanism of action of genes in diseases is of crucial importance. Gene-deficient mouse models are mainly achieved through gene editing techniques, among which the CRISPR / Cas9 system is one of the most commonly used tools at present; the CRISPR / Cas9 system uses specific RNA to guide the Cas9 protein to precisely cut the target DNA sequence, thereby achieving gene knockout, insertion or mutation; the advantage of this type of technology lies in its simple operation, high efficiency, and the ability to achieve conditional knockout, enabling researchers to study the function of genes in specific tissues or cells. Although the CRISPR / Cas9 technology has significant advantages in gene editing, there are still certain limitations in practical applications. For example, the efficiency and accuracy of gene editing are affected by various factors, including the design of gRNA, the expression level of Cas9 protein, and the characteristics of the target DNA sequence. In addition, unexpected mutations may occur during the gene editing process, such as insertions or deletions, which may affect the function of genes or lead to other unforeseen consequences.

[0005] The information disclosed in this background section is only used to deepen the understanding of the background of the present disclosure, and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] Constructing a gene-deficient mouse model is a complex and difficult process with great biological uncertainties. However, based on long-term experimental research, the present inventors found that by comprehensively adopting various strategies, such as optimizing the design of gRNA, increasing the expression level of Cas9 protein, using high-precision gene editing techniques, etc., the efficiency and accuracy of gene editing can be improved, and then an effective and high-quality TAGLN2 gene-deleted mouse with obvious phenotypic characteristics (abnormal in TAGLN2 functional regulation), genetic stability (the gene knockout state can be stably inherited in offspring), good basic health status (can grow and develop normally to meet experimental requirements), and good experimental repeatability (for mice of the same strain under different batches and different laboratory conditions, their gene knockout states and phenotypic characteristics have high consistency and repeatability).

[0007] Based on this, the present disclosure provides a TAGLN2 method for constructing a gene-deficient mouse model, specifically including the following steps: (1) Design and in vitro transcribe sgRNA targeting the TAGLN2 gene based on the CRISPR / Cas9 gene editing technology; (2) Co-microinject the sgRNA and Cas9 mRNA into mouse fertilized eggs; (3) Transplant the fertilized eggs that survived after injection in step (2) into the uterus of pseudopregnant female mice to breed F0 generation mice, and perform genotype identification on the F0 generation mice to screen positive F0 mice; (4) Mate the positive F0 mice with opposite-sex wild-type mice, and perform genotype identification on the offspring to obtain F1 generation heterozygous gene knockout mice; (5) Breed the F1 generation heterozygous gene knockout mice and perform genotype identification to obtain F2 generation homozygous gene knockout mice, which are the TAGLN2 gene-deficient mice; The sgRNA includes TAGLN2 the recognition sequence sgRNA-1 or sgRNA-2 upstream of the second exon of the gene, and TAGLN2 the recognition sequence sgRNA-3 or sgRNA-4 downstream of the fifth exon of the gene; Among them, the sequence of the sgRNA-1 is shown in SEQ ID NO.1; the sequence of the sgRNA-2 is shown in SEQ ID NO.2; the sequence of the sgRNA-3 is shown in SEQ ID NO.3; the sequence of the sgRNA-4 is shown in SEQ ID NO.4.

[0008] In some embodiments of the present disclosure, the PCR primer sequences for genotype identification are shown in SEQ ID NO.5~SEQ ID NO.8.

[0009] According to another aspect of the present disclosure, the TAGLN2 gene deletion mouse model constructed by the above method is applied to any one of the following (1)~(8): (1) Research on the mechanism of metabolic diseases or preparation / screening of drugs for metabolic diseases; (2) Screening or preparation of drugs for regulating carbohydrate metabolism, amino acid metabolism and / or lipid metabolism; (3) Screening or preparation of drugs for regulating ascorbic acid metabolism, aldol ester metabolism and / or butyric acid metabolism; (4) Screening or preparation of drugs for regulating the interconversion of pentose and glucuronic acid; (5) Screening or preparation of drugs for regulating glycolysis and / or glucose production; (6) Screening or preparation of drugs for regulating arachidonic acid metabolism, fatty acid biosynthesis and / or steroid hormone biosynthesis; (7) Screening or preparation of drugs for treating cancer; (8) Screening or preparation of drugs for regulating central carbon metabolism, amino acid biosynthesis, glyoxylate metabolism, dicarboxylate metabolism, carbon metabolism, citric acid cycle, pyruvate metabolism and / or β-alanine metabolism.

[0010] One or more technical solutions provided in the embodiments of the present disclosure have at least any one of the following technical effects or advantages: Based on the CRISPR / Cas9 gene editing technology, the gRNA is used to guide the Cas9 protein to TAGLN2 perform cleavage at specific sites upstream of the second exon and downstream of the fifth exon of the TAGLN2 gene, causing double-strand breaks in the DNA. Subsequently, the cell repairs the cleavage site using non-homologous end joining, thereby achieving TAGLN2 effective knockout of the gene. The mouse model has obvious phenotypic characteristics, genetic stability, good health, and good experimental repeatability. This mouse model can provide a basis for studying the Molecular mechanism of the gene provides help for the study of the pathogenesis of metabolic diseases, drug development, and evaluation of effectiveness and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of gene knockout in an embodiment of the present disclosure TAGLN2

[0012] Figure 2 PCR identification results of the genotypes of F0 generation mice in an embodiment of the present disclosure TAGLN2

[0013] Figure 3 PCR identification results of the genotypes of F1 generation mice in an embodiment of the present disclosure TAGLN2

[0014] Figure 4 PCR identification results of the genotypes of F2 generation mice in an embodiment of the present disclosure TAGLN2

[0015] Figure 5 KEGG analysis results of RNA-seq of retinas of knockout mice and control mice in an embodiment of the present disclosure TAGLN2

[0016] Figure 6 Metabolome analysis results of retinas of knockout mice and control mice in an embodiment of the present disclosure TAGLN2 DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] In order to better understand the technical solutions of the present invention, the above technical solutions will be described in detail below in conjunction with the accompanying drawings of the specification and specific embodiments.

[0018] In the following embodiments, the instrument equipment involved is all conventional instrument equipment unless otherwise specified; the reagents involved are all commercially available conventional reagents unless otherwise specified; the detection methods involved are all conventional methods unless otherwise specified. ​​​​

[0019] Example 1 TAGLN2 Construction of knockout mice Using the CRISPR / Cas9 gene editing technology, the Cas9 protein was guided by gRNA to perform cleavage at specific sites upstream of the second exon and downstream of the fifth exon of the TAGLN2 gene (the cleavage sites are between the third and fourth nucleotides upstream of the PAM sequence near the 3' end of the sgRNA), causing double-strand breaks in the DNA, and then using non-homologous end joining to repair the cleavage sites ( Figure 1 ).

[0020] 1. Design and synthesize sgRNA, the sequences are as follows: sgRNA-1: 5`-CCAGGCGTCATTCTAGACAC-3` (SEQ ID NO.1), PAM: AGG; sgRNA-2: 5`-CTGTCAAGCTTCATCACTCC-3` (SEQ ID NO.2), PAM: AGG; sgRNA-3: 5`-CAATTATCAGGCCCTGCCAG-3` (SEQ ID NO.3), PAM: TGG; sgRNA-4: 5`-AGAGGGGAGTTCAGCAATCC-3` (SEQ ID NO.4), PAM: AGG.

[0021] 2. Superovulation treatment of mice and isolation of fertilized eggs: Take 4-week-old C57BL / 6J female mice, intraperitoneally inject 10 IU of pregnant mare serum gonadotropin (PMSG). 48 h after injecting PMSG, intraperitoneally inject 10 IU of pregnant mare serum gonadotropin (PMSG) again to promote the ovulation of developed and mature follicles. After injecting hCG, immediately cage one female mouse with one male mouse. Check for vaginal plugs the next morning. Anesthetize and sacrifice the C57 / BL6J female mice with vaginal plugs, isolate the oviducts, aspirate PBS solution with a syringe and rinse the oviducts under a microscope to allow the fertilized eggs to flow out of the oviducts. Transfer the fertilized eggs to 1 mg / mL hyaluronidase (Sigma, 37326-33-3) for digestion for 2 min, wash 3 times with M2 medium (Sigma, M7167), and then place the fertilized eggs in M16 medium (Sigma, M7292) and culture in an incubator at 37°C and 5% CO2 for 2 h.

[0022] 3. Preparation of pseudopregnant female mice: Male mice at 8 - 12 weeks of age were ligated, and their vas deferens were cut so that they could not transport semen but still had the ability to mate. Virgin female mice at 8 - 12 weeks of age were caged with the vasectomized male mice, and the female mice on the 4th day after detecting vaginal plugs were used as pseudopregnant female mice.

[0023] 4. Microinjection: Cas9 mRNA, sgRNA - 1, and sgRNA - 3 were mixed and co - microinjected into mouse fertilized eggs. Among them, the injection concentration of Cas9 mRNA (ThermoFisher, A29378) was 50 ng / μl, the injection concentration of sgRNA - 1 was 20 ng / μl, and the injection concentration of sgRNA - 3 was 20 ng / μl; Cas9 mRNA, sgRNA - 2, and sgRNA - 4 were co - microinjected into other mouse fertilized eggs. Among them, the injection concentration of Cas9 mRNA was 50 ng / μl, the injection concentration of sgRNA - 2 was 20 ng / μl, and the injection concentration of sgRNA - 4 was 20 ng / μl. The fertilized eggs after microinjection were transferred to M16 medium and cultured overnight in an incubator at 37°C and 5% CO2.

[0024] 5. Generation and screening of F0 - generation mice: The surviving fertilized eggs after injection were transplanted into the uterus of pseudopregnant female mice to breed F0 - generation mice. At 9 - 11 days after the mice were born, 2 - mm tail tissues of the mice were cut, and genomic DNA of the mice was extracted using a DNA extraction kit. PCR genotyping was performed on F0 - generation mice to screen positive F0 - generation mice. When there was a band in the PCR amplification product using F1 / R1 primers and no band in the PCR amplification product using F2 / R2 primers, F0 - generation homozygous knockout mice were obtained. TAGLN2 The results of genotype identification are as Figure 2 shown. Lanes 12 - 17 are F0 - generation homozygous TAGLN2 knockout mice (positive F0 - generation mice).

[0025] 6. Generation and screening of F1 - generation mice: Positive F0 - generation mice were mated with opposite - sex wild - type mice, and PCR genotyping was performed on the offspring. When there was a band in the PCR amplification product using F1 / R1 primers and also a band in the PCR amplification product using F2 / R2 primers, F1 - generation heterozygous gene - knockout mice were obtained. The results of genotype identification are as Figure 3 shown. Lanes 31 - 33 and 59 - 61 are F1 - generation heterozygous mice.

[0026] 7. Construction and screening of F2 generation mice: Breed male and female F1 generation heterozygous mice by inbreeding, and identify the genotypes of the offspring by PCR. When there is no band in the PCR amplification product using F1 / R1 primers and there is a band in the PCR amplification product using F2 / R2 primers, the genotype of the detected mouse is wild type; when there is a band in the PCR amplification product using F1 / R1 primers and there is also a band in the PCR amplification product using F2 / R2 primers, the genotype of the detected mouse is heterozygous; when there is a band in the PCR amplification product using F1 / R1 primers and there is no band in the PCR amplification product using F2 / R2 primers, it is the F2 generation homozygous TAGLN2 gene knockout mouse. The PCR identification results of the mouse genotypes are as Figure 4 shown. Lanes 9, 13, and 16 are wild-type mice, lanes 4, 11, 12, 14, and 18 are F2 generation heterozygous mice, and lanes 1-3, 5-8, 10, 15, and 17 are F2 generation homozygous TAGLN2 knockout mice.

[0027] In the above steps, a tissue genomic DNA extraction kit (DP304, column type, Tiangen Biochemical Technology Co., Ltd.) was used to extract the genomic DNA of mouse tissues. The specific steps are as follows: (1) At 9-12 days after the mouse is born, cut 2 mm of the tail tissue of the mouse, use a tissue grinder to grind the tissue into a cell suspension, centrifuge at 10000 rpm for 1 min, discard the supernatant, add 200 μl of buffer GA, and shake until the precipitate is completely suspended.

[0028] (2) Add 20 μl of Proteinase K solution, mix well, incubate and digest at 56 °C, invert and mix the sample 2-3 times until the tissue cells are lysed.

[0029] (3) Add 200 μl of buffer GB, mix well by inverting thoroughly, and place at 70 °C for 10 min until the solution becomes clear.

[0030] (4) Add 200 μl of absolute ethanol and shake well for 15 s.

[0031] (5) Add the solution obtained in step (4) to adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 30 s, discard the waste liquid, and put the adsorption column back into the collection tube.

[0032] (6) Add 500 μl of buffer GD to adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and put adsorption column CB3 back into the collection tube.

[0033] (7) Add 600 μl of washing buffer PW to adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column back into the collection tube.

[0034] (8) Repeat step (7).

[0035] (9) Place adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste liquid, and leave adsorption column CB3 at room temperature for 5 min.

[0036] (10) Transfer adsorption column CB3 into a clean centrifuge tube, add 50 μl of elution buffer TE, leave at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0037] PCR genotype identification: TAKARA Premix Ex Taq™ Hot Start Version PCR amplification enzyme (TAKARA, RR030) was used for PCR genotype identification. The specific steps are as follows: Using mouse genomic DNA as a template, configure the PCR reaction system (Table 1). Among them, the PCR primer sequences are as shown in SEQ ID NO.5 - SEQ ID NO.8.

[0038] 。

[0039] Briefly mix and centrifuge each reaction solution in the above reaction system, and perform PCR amplification reaction. The PCR reaction parameters are: pre-denaturation at 95 °C for 5 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 30 cycles; extension at 72 °C for 10 min.

[0040] Configure the PCR product into an electrophoresis loading system (Table 2), electrophorese the PCR product on a 1% agarose gel at 120 V for 30 min, and observe the banding pattern to determine its genotype.

[0041] 。

[0042] Example 2 TAGLN2 Metabolic study of gene knockout mice Take 20 8-week-old male gene knockout and wild-type mice constructed in Example 1 each, isolate mouse retinal tissues for transcriptome high-throughput sequencing and metabolome analysis. TAGLN2

[0043] 1. RNA extraction Extract mouse retinal tissue RNA using TRIZOL Reagent (Invitrogen, 15596026CN): (1)After anesthetizing the mice, decapitate them to sacrifice. Take out the eyeballs, quickly separate the retinas and put them into EP tubes. Add 1 ml of Trizol and use a tissue grinder to grind the tissues into homogenates.

[0044] (2)Lyse and digest at room temperature for 5 min, centrifuge at 12,000 g for 10 min at 4 °C, and transfer the supernatant to another EP tube.

[0045] (3)Add 200 μl of chloroform, tightly cap the EP tube, shake vigorously. After the solution is fully emulsified and turns milky yellow, let it stand at room temperature for 3 - 5 min, and centrifuge at 12000 g at 4 °C for 15 min.

[0046] (4)Carefully take out the EP tube from the centrifuge. At this time, the homogenate is divided into three layers. Gently aspirate the supernatant of the top layer and transfer it to another new EP tube.

[0047] (5)Add 500 μl of isopropanol, invert up and down to mix well, let it stand at room temperature for 10 min, and centrifuge at 12000 g at 4 °C for 10 min.

[0048] (6)Carefully discard the supernatant, add 1 mL of 75% ethanol, gently shake the centrifuge tube to suspend the precipitate, centrifuge at 8000 g at 4 °C for 5 min, then carefully discard the ethanol, dry the precipitate at room temperature for 2 - 5 min, and add 50 μL of RNase-free water to dissolve the precipitate.

[0049] 2. Transcriptome sequencing (1)For the total RNA extracted from the retinal tissue, detect the RNA concentration and quality through the Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, USA) systems. If the quality inspection is qualified, construct an mRNA library.

[0050] (2)Use the BGI-seq500 platform for transcriptome high-throughput sequencing. The original sequencing data are processed by SOAPnuke to remove low-quality, adapter-polluted reads and reads with an unknown base N content > 5% to obtain high-quality sequences.

[0051] (3) Align the high-quality sequences to the NCBI reference genome sequence (GCF_000001635.26_GRCm38.p6) using HISAT (v2.0.4), and align the high-quality sequences to the NCBI reference gene sequence (GCF_000001635.26) using Bowtie2 (v2.2.5) to obtain the alignment results.

[0052] (4) Calculate the Pearson correlation coefficient of all gene expression levels between every two samples to reflect the correlation of gene expression among samples.

[0053] (5) Calculate the gene expression levels using RSEM (v1.2.12), and perform differential expression gene analysis using the DESeq2 (v1.4.5) software. The significance criterion for difference is P <0.05.

[0054] (6) Use the phyper function in R software to perform KEGG (Kyoto Encyclopedia of Genes and Genomes, https: / / www.kegg.jp / ) enrichment analysis. The significance criterion for enrichment is P <0.05.

[0055] The detection results are as Figure 5 shown. After transcriptome high-throughput sequencing, KEGG analysis was performed, and 18 pathways were significantly enriched ( Q <0.05), mainly enriched in carbohydrate metabolism pathways such as ascorbate and aldarate metabolism, butanoate metabolism, pentose and glucuronate interconversions, and glycolysis / gluconeogenesis, as well as lipid metabolism pathways such as arachidonic acid metabolism, fatty acid and steroid hormone biosynthesis, indicating that TAGLN2 the carbohydrate metabolism and lipid metabolism capabilities of the gene-deficient mouse model are enhanced, and it can be applied to the development and screening of drugs for regulating carbohydrate metabolism or lipid metabolism.

[0056] 3. Metabolomics analysis The metabolomics analysis process mainly includes steps such as sample preparation, metabolite separation and detection, data analysis, and screening of biomarkers. The specific process is as follows: (1) Sample preparation: Take appropriate amounts of samples and QC, add 140 ul of 50% water / methanol solution, use ultrasonic wave to break the tissue, centrifuge at 12000 g at 4 °C for 10 min, and take the supernatant; Standard curve preparation: Take the HM400 mixed standard for gradient dilution to prepare the standard curve; Derivatize the samples, QC, and standard curve; Dilute with the HM400 diluent, centrifuge at 18000 g, 4 °C for 10 min, and take the supernatant for LC-MS analysis.

[0057] (2)Metabolite separation and detection: Waters 2777C UPLC (Waters, USA) was used in tandem with a Q ExactiveHF high-resolution mass spectrometer (Thermo Fisher Scientific, USA) for the UPLC-MS separation and detection of metabolites.

[0058] (3)Data processing and analysis: The detected metabolite data was further preprocessed using metaX software to obtain compounds and quantitative values for formal analysis, including baseline correction, peak detection, normalization, etc. Then, chemometric methods such as principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), etc. were used for data dimensionality reduction and pattern recognition. The data quality was evaluated through the repeatability of QC sample detection. A data matrix containing metabolite identification results and quantitative results was obtained using the quantitative software skyline.

[0059] (4)Biomarker screening and validation: Information annotation of the metabolite identification results was performed through KEGG and the Human Metabolome Database (HMDB), including KEGG ID, HMDB ID, category, and the KEGG metabolic pathways involved. Through these analysis methods, differences in metabolites among different samples or different biological states could be found. According to the data analysis results, metabolites related to specific biological states were screened out as biomarkers. Then, independent sample sets were used for validation to evaluate the specificity and sensitivity of the biomarkers.

[0060] (5)Biological interpretation: Biological interpretation of the screened biomarkers was carried out to explore their associations with specific biological processes or diseases.

[0061] The detection results are as Figure 6 shown. Metabolome analysis significantly enriched 10 pathways ( Q <0.05), including central carbon metabolism in cancer, biosynthesis of amino acids, glyoxylate and dicarboxylate metabolism, carbon metabolism, citric acid cycle, pyruvate metabolism, glycolysis / gluconeogenesis, and β-alanine metabolism, indicating that TAGLN2 the carbohydrate metabolism and amino acid metabolism capabilities of the gene-deficient mouse model were enhanced, and it could be applied in the research and screening of drugs for regulating carbohydrate metabolism and amino acid metabolism capabilities.

[0062] Although some preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0063] Obviously, those skilled in the art can make various modifications and variations to the present disclosure without departing from the spirit and scope of its inventive concept. Thus, if these modifications and variations to the present disclosure fall within the scope of the claims of this application and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A TAGLN2 The method for constructing a gene-deficient mouse model comprises the following steps: (1) Design and in vitro transcription targeting based on CRISPR / Cas9 gene editing technology TAGLN2 sgRNA of the gene; (2) simultaneously microinjecting the sgRNA and Cas9 mRNA into mouse fertilized eggs; (3) transplanting the mouse fertilized eggs described in step (2) into the uterus of a pseudo-pregnant female mouse to breed F0 generation mice, performing genotyping on the F0 generation mice, and screening positive F0 mice; (4) mating the positive F0 mice with wild-type mice of the opposite sex, identifying the genotype of the offspring, and obtaining F1 heterozygous gene knockout mice; (5) breeding the F1 generation heterozygous gene knockout mice and performing genotyping to obtain F2 generation homozygous gene knockout mice, namely the TAGLN2 Gene-deficient mice; The sgRNA includes TAGLN2 The recognition sequence sgRNA-1 or sgRNA-2 upstream of the second exon of the gene, and TAGLN2 recognition sequence sgRNA-3 or sgRNA-4 downstream of exon 5 of the gene; Among them, the sequence of the sgRNA-1 is shown as SEQ ID NO.1; the sequence of the sgRNA-2 is shown as SEQ ID NO.2; the sequence of the sgRNA-3 is shown as SEQ ID NO.3; and the sequence of the sgRNA-4 is shown as SEQ ID NO.

4.

2. The construction method according to claim 1, characterized in that: The PCR primer sequences for genotype identification are shown in SEQ ID NO.5 to SEQ ID NO.

8.

3. The method according to claim 1 TAGLN2 Application of gene-deficient mouse models in any of the following (1) to (8): (1) Research on the mechanism of action of metabolic diseases or preparation / screening of drugs for metabolic diseases; (2) Screening or preparing drugs that regulate carbohydrate metabolism, amino acid metabolism and / or lipid metabolism; (3) Screening or preparing drugs that regulate ascorbic acid metabolism, aldehyde ester metabolism and / or butyrate metabolism; (4) Screening or preparing drugs that regulate the interconversion between pentose and glucuronic acid; (5) Screening or preparing drugs that regulate glycolysis and / or glucose production; (6) Screening or preparing drugs that regulate arachidonic acid metabolism, fatty acid biosynthesis and / or steroid hormone biosynthesis; (7) Screening or preparing drugs for treating cancer; (8) Screening or preparing drugs that regulate central carbon metabolism, amino acid biosynthesis, glyoxylate metabolism, dicarboxylate metabolism, carbon metabolism, citric acid cycle, pyruvate metabolism and / or β-alanine metabolism.

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